Freeze-dried hepatitis A attenuated live vaccine and its stabilizer

ABSTRACT

The present invention relates to hepatitis A vaccine, especially to a lyophilized attenuated hepatitis A vaccine which can be stored at ambient temperature for extended periods of time, and to a method for producing the same. The present invention further relates to a stabilizer for lyophilized live vaccine and its use in improving thermostability of lyophilized live vaccine during lyophilization processing and storage period after lyophilization.

FIELD OF THE INVENTION

The present invention generally relates to attenuated hepatitis Avaccine, and more particularly to a stabilized lyophilized livehepatitis. A vaccine formulation which can be preserved at ambienttemperature for extended periods of time, to eliminate the pressuresfrom transportation, storage and usage of the vaccine without loss ofinfectivity titers of the vaccine. The present invention further relatesto a stabilizer for live lyophilized vaccine and its use in producingstabilized lyophilized live vaccine formulations.

BACKGROUND OF THE INVENTION

Hepatitis A is a worldwide distributive acute disease caused byinfection with hepatitis A virus (HAV) which is a picornavirus closelyrelated to the poliovirus. Infection is spread by the fecal/oral routeand consequently the disease in endemic in areas where hygiene andsanitation standards are lower. Recent reports on epidemical survey showthat in developing countries including China, there are as many as 4million hepatitis A cases per year. There is frequently large-scaleoutbreak and rapid spread in certain regions with poor social andeconomic status, especially after various disasters. In these countriesor regions, as the high incidence of hepatitis A, some serious publichealth and social problems have been encountered. On the other hand, inthe United States and other developed countries, hepatitis A accountingfor approximately 150,000 cases, that is approximately 25% of allclinical hepatitis cases.

Therefore, to successfully immunize against hepatitis A in developingcountries as well as in developed countries, it is necessary tovaccinate the entire people, especially entire pediatric populations. Sothere will be an increasing need for hepatitis A vaccine.

An effective vaccine would be useful for active immunization ofpopulations at high risk. Generally, there are four types of vaccinesused for inducing a specific neutralizing antibody against challengewith virus or bacteria: live vaccine, inactivated vaccine, subunitvaccine (component vaccine), and recombinant vaccine. In these vaccines,the live attenuated vaccine could elicit a stronger protective responsethan others, and could have a significant impact on the eradication ofthe diseases.

U.S. Pat. Nos. 4,532,215 and 4,636,469 described, respectively a strainof wild-type HAV, designated HM-175, initially isolated from the facesof a patient, and adapted to passage in vitro in African green monkeykidney culture cell and methods for obtaining a vaccine by serialpassaging. Also, CN Patent Nos. 89106580.6 and 92114998 disclose thepreparation of attenuated HAV designated H₂-and L-A-I, respectively.

With regard to live attenuated hepatitis A vaccine, it is worthmentioning the live HAV vaccine based on strain CR*326F (Merck & Co.Inc.), which is under preclinical trials, and the vaccines based onstrain H₂ and L-A-I, respectively, have been licensed for practical useand industrial-scale production in China. Clinical serological studiesdemonstrated that these live attenuated hepatitis A vaccines, especiallythe vaccine prepared from L-A-I strain of HAV (produced by ChangchunInstitute of Biological Products Ministry of Public Health, Changchun,China) evoked high titers of antibody response, in most volunteersreceiving the vaccine, after only one dose and no systemic complainswere present immediately after vaccination or during long-term follow-up(see CN Patent No. 92214998).

However, all of the live hepatitis A vaccines used so far are in theform of aqueous suspensions. One of the main disadvantages of liveattenuated vaccine is having unsatisfactory theremo-stability, even inthe situation of lyophilization at ambient temperature, hence it must bestored and transported in a frozen state and used soon after thawing toinsure effective vaccination. Hepatitis A virus, as well as measlesvirus is unsatisfactory in both storage stability and heat resistance.For example, live attenuated hepatitis A virus survives only for about 7days at a temperature of 2-8° C., and storage-term duration is onlyabout 3-6 months. Therefore, transportation and storage of these vaccinepreparations must be completed at a reduced temperature (e.g., −20° C.or lower), referred to as “cold chain.” As a direct result, theincreases in production and transportation cost and user's expense areunavoidable, especially in developing countries and tropical andsemitropical areas. This cost would be an obstacle to implementation ofthe worldwide Expanded Program on Immunization (EPI) founded by WorldHealth Organization (WHO).

For the reasons as described above, eradication of hepatitis A willdepend on the ability to provide hepatitis. A vaccine formulationshaving improved thermo-stability. Accordingly, there remains a distinctneed in the art for live hepatitis vaccine formulations with enhancedstorage stability and heat resistance during and after lyophilization.

SUMMARY OF THE INVENTION

In view of the problems mentioned above, the present inventors haveperformed intensive experiments during their production practices toovercome these problems and to provide a lyophilized live hepatitis Avaccine with increased thermo-resistance and storage stability. Thepresent inventors have surprisingly found that when a stabilizersolution is added to the vaccine stock suspension prepared by adisclosed method (for example as described in CN Patent No. 92114998),and lyophilized the hepatitis A vaccine formulation comprising, as avirus component, an attenuated live hepatitis A virus and a stabilizer,the storage-term of the hepatitis A virus containing lyophilized vaccineis extended 3 times longer than non-treated stock viral suspension.Therefore, the “cold chain” pressure and user's expense is describedgreatly thereby increasing the ability for low-cost, widespread use.

It is one object of the present invention to provide a stabilizedlyophilized hepatitis A live vaccine formulation comprising aprophylactically effective viral titers of live attenuated hepatitis Avirus and a stabilizer which can be preserved at ambient temperature forextended periods of time, so that the “cold chain” pressures fromtransportation, storage and usage of the vaccine can be reduced oreliminated without loss of infective titers of the vaccine, therebygreatly decreasing the expense and relevant cost to ensure effectivewidespread vaccination against hepatitis A.

In a preferred embodiment of the present invention, said stocksuspension of live attenuated hepatitis A virus is prepared by disclosedmethod for the wild-type HAV, stain L-A-I.

In a another preferred embodiment of the present invention, saidstabilizer for lyophilized live hepatitis A virus is composed ofgelatin, trehalose, one or two amino acid selected from the groupconsisting of glutamic acid, aspartic acid, arginine, lysine or alkalimetal salts thereof, ascorbic acid, urea, mannitol or sorbitol or bothof them, and inositol.

According to a further preferred embodiment of the present invention,the stabilizer for lyophilized live virus vaccine contains human serumalbumin.

In a further preferred embodiment of the stabilizer according to thepresent invention, the stabilizer for the lyophilized live virus isessentially composed of from 0 to 20 grams per liter of human serumalbumin, from 5 to 10 grams per liter of gelatin, from 50 to 100 gramsper liter of trehalose, about 7.5 to 15 grams per liter of sodiumglutamate, from 0.5 to 5.5 grams per liter of ascorbic acid, from 5 to28 grams per liter of urea, from 2 to 10 grams per liter of mannitol orsorbitol or a mixture, and from 4 to 10 grams per liter of inositol.

It is another object of the present invention to provide a method ofpreparing stabilized lyophilized live hepatitis A vaccine formulation asabove, comprising:

-   -   (a) providing a stock suspension of attenuated live Hepatitis A        virus;    -   (b) adding a stabilizer solution to the stock suspension of        step (a) at the ratio 1:1 (v/v) to obtain a live vaccine        formulation comprising prophylactically effective viral titers        of live attenuated hepatitis A virus and a stabilizer for        attenuated live virus, therein said stabilizer comprises        gelatin, trehalose, one or two amino acid selected from the        group consisting of glutamic acid, aspartic acid, arginine,        lysine or alkali metal salts thereof, ascorbic acid, urea,        mannitol or sorbitol or both of them, and inositol;    -   (c) lyophilizing said vaccine formulation obtained from the step        (b).

According to a preferred embodiment of this object of the invention, thelyophilization step comprises precooling the vaccine formulation toabout −20 to −50° C. over about 3 to 6 hours, and then drying the livevaccine formulation by gradually increasing the temperature from −38 to35° C. in a lyophilizer.

It is a further object of the present invention to provide a stabilizerfor lyophilized live virus, wherein said stabilizer is essentiallycomposed of gelatin, trehalose, one or two amino acid selected from thegroup consisting of glutamic acid, aspartic acid, arginine, lysine oralkali metal salts thereof, ascorbic acid, urea, mannitol or sorbitol orboth of them, and inositol.

According to a preferred embodiment of the present invention, saidstabilizer is essentially composed of from 0 to 20 grams per titer ofhuman serum albumin, from 5 to 10 grams per liter of gelatin, from 50 to100 grams per liter of trehalose, from 7.5 to 15 grams per liter ofsodium glutamate, from 0.5 to 5.5 grams per liter of ascorbic acid, from5 to 28 grams per liter of urea, from 2 to 10 grams per liter ofmannitol or sorbital or a mixture of them, and from 4 to 10 gram perliter of inositol.

According to a further preferred embodiment of the present invention,the stabilizer for lyophilized live virus vaccine can contain humanserum albumin.

According to preferred embodiment of the present invention, saidstabilizer is not only suitable for stabilizing lyophilized hepatitis Alive virus, but also used for stabilizing viruses selected from thegroup consisting of the genus Enterovirus, the genes Paramyxovirus, thegenus Arbovirus, and the genus Herpesvirus against heat inactivationduring the period of lyophilization and the period of storage andtransportation post-lyophilization to ensure thermo-stability of thelyophilized live vaccine thereby to improve vaccination efficacy forsusceptible populations.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to lyophilized live vaccineformulations having a increased thermo-stability. Essentially, thevaccine formulations of the present invention are mixtures of viruscomponent and stabilizer components, wherein the virus componentcomprises hepatitis A virus or at least one member selected from thegenuses Enterovirus, Pavamyxovirus, Arbovirus and Herpesvirus, and thestabilizer components comprise gelatin, trehalose, one or two aminoacids selected from the group consisting of glutamic acid, asparticacid, arginine, lysine or alkali metal salts thereof, ascorbic acid,urea, mannitol and/or sorbitol, and inositol. The vaccine stabilizeraccording to the present invention can contain human serum albumin (HSA)to prevent any undesirable enzymolysis of the virus. Upon mixing the twocomponents in a suitable ratio, the result is a virus formulation whichcontains from about 0 to 20 grams per liter of HSA, from 5 to 10 gramsper liter of gelatin, from 50 to 100 grams per liter of trehalose, from7.5 to 15 grams per liter of amino acid or alkali metal salts thereof,from 0.5 to 5.5 grams per liter of ascorbic acid, from 5 to 28 grams perliter of urea, about 2 to 10 grams per liter of mannitol or sorbitol ora mixture of them, and from 4 to 10 grams per liter of inositol.

With respect to a attenuated hepatitis A virus, for example, the HAVstock suspension used for the purpose of the present invention could beprepared from the wide-type strain L-A-I by the HAV cell-cultureadaption and attenuation method described in CN Patent No. 92114998.Briefly, the method comprises cultivating human diploid fibroblast cellsin a suitable nutrient medium, e.g., Eagle's minimal essential medium(MEM), containing 10-15% fetal calf serum (FCS) in a roller bottle at37° C. for 5-8 days. When confluent cell monolayers are formed, thecultured medium is discarded from the culturing vessel and the cells arewashed with the same medium or PBS 3 to 5 times. The cells areinoculated with a seed virus of hepatitis A virus L-A-I derived fromhuman feces and purified by the method of described in Example 1 of CNPatent No. 92114998. The cells are then cultivated in nutrient medium atabout 34-36° C. for 3 to 4 weeks After completion of cultivation, thenutrient medium is changed to medium 199 with or without phenol red, andcells are cultivated at 34 to 36° C. for a additional 4 to 6 days in acell roller. After harvesting, the cells are sonificated 3 times, thenthe cell debris is removed by centrifugation and the resultantsupernatant is collected to obtain the desired stock suspension of thevirus.

The present invention further provides a stabilizer advantageously usedto stabilize a live vaccine, and to protect attenuated live virusagainst heat-inactivation at ambient temperature for a extended periodsof time, essentially composed of human serum albumin and/or gelatin,trehalose, one or two amino acids selected from the group consisting ofglutamic acid, aspartic acid, arginine, lysine or alkali metal saltsthereof, ascorbic acid, urea, mannitol and/or sorbitol, and inositol. Ina particularly preferred embodiment, the present invention provides astabilizer containing about 0-20 g/L of HSA, about 5-10 g/L of gelatin,about 50-100 g/L of trehalose, about 7.5-15 g/L of sodium glutamate,about 0.5-5.5 g/L of ascorbic acid, about 5-28 g/L of urea, about 2-10g/L of mannitol and/or sorbitol, and 4-10 g/L of insoitol.

Hepatitis A virus is a small picornavirus with no outer envelope orother lipids. Like the majority of live enteroviruses, hepatitis A viruspresented in form of aqueous suspension will rapidly lose the ability ofreplication, propagation, and/or infectious potency. In the absence of asuitable stabilizer, effective protection against hepatitis A infectionis extremely susceptable to heat inactivation of the virus. Thuseradication of hepatitis A and other epidemic caused by infection withvirus will depend on the ability to assure cold storage andtransportation of virus vaccine. However, according to the presentinvention, this problem has been circumvented by using vaccineformulations with improved stability characteristics.

Vaccine stabilizers are well known in the art as chemical compoundsadded to vaccine formulations to enhance vaccine stability duringperiods of low temperature storage, lyophilization processing, orstorage post-lyophilization. As described above, the stabilizer aqueoussolutions used for formulating and stabilizing the live vaccine of thepresent invention are preferably composed of a high molecular weighstructural additive, a disaccharide, a sugar, alcohol and water. Theaqueous solution also includes one or two amino acids and a bufferingcomponent. The combination of these components act to preserve thesurvival and activity of the virus upon freezing and lyophilization anda long storage period subsequent to lyophilization.

It is well known that high molecular weight structural additives aid inpreventing viral aggregation during freezing, and provide structural andnutritional support in the lyophilized or dried state. Within thecontext of the present invention, the preferred high molecular weightstructural additives are human serum albumin and/or gelatin. The aminoacids, sugars, and alcohols function to further preserve viralinfectivity upon cooling and thawing of the aqueous suspension. Inaddition, these components function to preserve viral infectivity duringsublimation of cooled aqueous virus suspensions during lyophilizationand in lyophilized state, and contribute some buffering ability. Thepreferred amino acids are arginine and glutamate, and the preferredsugar alcohols are mannitol, sorbitol and inositol. Trehalose is thepreferred disaccharide used in the stabilizer aqueous solution and couldbe extremely beneficial for stabilizing the protein structure of thevirus to increase heat-resistance and for restoring the potency of thevirus after dehydration. Urea and ascorbic acid play an important partin stabilizing the hydration state or in maintaining osmotic balanceduring dehydration period. The buffering component acts to buffer theformulation by maintaining a relatively constant pH which is preferablyabout 7.0. The preferred buffer is balanced salt solution or PBS usedfor dissolving the chemical compounds disclosed above.

The components are added in increasing amounts to generate vaccinestabilizer to combine with viral stock suspensions to generate vaccineformulations for lyophilization that have an increased increase inthermo-stability. The preferred component ranges disclosed in thisspecification allow for generation of vaccine formulations which, amongother characteristics, exhibit improved thermo-stability over vaccineformulations known in the art.

The stabilizer of live attenuated vaccine can be formulated byconventional methods, for example, by mixing each of the components in asuitable vessel, except for preheating the mixture solution oftrehalose, gelatin, urea, mannitol and/or sorbitol at about 37° C. for24 to 28 hours before adding HSA thereto. After 0.5 to 2 hours, mixingthe resultant stabilizer is mixed with viral stock at about 1:1 (v/v)ratio.

It is noteworthy that the ranges of virus stabilizer and final vaccineformulation are presented on a gram per liter basis of the final vaccineformulation. One of ordinary skill in the art will be well aware thatchanging volume ratio of stabilizer to vaccine may be applied topractice the claimed invention, which in turn will require changes tothe concentration of stabilizer components. Therefore, the invention isnot only limited to the specified 1:1 stabilizer/virus combination togenerate the final vaccine formulation for lyophilization.

After the vaccine is formulated with stabilizer and viral stock, theresultant aqueous suspension should be dried by lyophilization. Briefly,the lyophilization cycles involves the steps of precooling the aqueoussuspension below the gas transition temperature or below the eutecticpoint temperature (below −30° C.) of the aqueous suspension for 3 to 6hours, and then removing water from the cooled suspension by sublimationto form a lyophilized virus. Within one preferred embodiment, aliquotsof the formulated attenuated live virus are placed into a refrigeratedchamber attached to a freeze dryer. A multistep freeze drying procedureis used in lyophilizing the formulated live vaccine. The temperature isthen gradually increased from about −38° C. to about 35° C. over aperiod of 10 to 20 hours.

In order to demonstrate improvement in thermal stability and storagestability of live vaccine, the present invention is exemplified by viralpotencies, for example for detecting viral titers pre- andpost-lyophilization of hepatitis A vaccine, and to observe theeffectiveness of a stabilizer on the storage stability of live vaccine.The results show that the stabilizer included in the vaccineformulations of the present invention at a concentration sufficient tostabilize the live virus vaccine against heat inactivation remarkablyimproved thermal stability of the virus which had been lyophilized andincubated at 37° C. for one week as measured by the log CCID₅₀, ascompared with lyophilized control vaccine formulation which is absent ofstabilizer.

Further, the present inventors in a comparison experiment found that asimilar result can be observed when a stabilizer solution, which doesnot contain the human serum albumin (HSA) component, is used for thelive virus vaccine if the lyophilization cycle parameters could besuitably adjusted.

The following examples are provided to illustrate the present invention.It is to be understood, however, that the examples are not to limit thescope of the present invention.

EXAMPLE 1

Preparation of stabilizer (I) in accordance with the present inventionfor lyophilized live virus:

Following components are utilized for formulating the stabilizersolution (I):

Component Amount (g/L) Human Serum Albumin 10.0 Gelatin  5.5 Trehalose65.0 Sodium Glutamate 10.0 Urea 20.0 Ascorbic Acid  5.5 Sorbitol  6.6Inositol  7.5300 ml of distilled water is added to 5.5 g of purified gelatin, and theresultant mixture is heated for 40 minutes in an autoclave (116° C.) toobtain a solution. The resulting solution is cooled to a temperature ofabout 30-35° C., and 10.0 g of HSA sterilized by ultrafiltration, isadded. In according with the formulation as above, corresponding amountof trehalose, sodium glutamate, urea, ascorbic acid, sorbitol andinositol are added in this order, to 500 ml of distilled water andthoroughly mixed. The resultant solution is heated for 24 hours at 37°C. After cooling the solution to ambient temperature (about 22-26° C.),the solution is placed into gelatin-HAS solution and mixed thoroughly.Subsequently, distilled water is added to bring the total volume to 1000ml. The pH of the resulting mixture solution is adjusted to about 7.0 by0.1 N HCL, and is subjected to filtration sterilization once again, toobtain a stabilizer solution (I) which could be used for stabilizinglive virus, to be lyophilized and stored.

EXAMPLE 2

Preparation of stabilizer (II) for lyophilized live vaccine:

The following components are utilized for formulating the stabilizersolution (II) in accordance with the present invention, by a proceduresimilar to that described in Example 1.

Component Amount (g/L) Gelatin  8.5 Trehalose 75.0 Urea 15.5 L-arginine10.1 Ascorbic Acid  3.0 Sorbitol  5.0 Mannitol  5.0 Inositol  4.0

HSA is eliminated as the component because it is very expensive and maycause virus contamination derived from collected blood sources.Furthermore, the sodium glutamate is replaced by arginine or alkalimetal salt thereof, and a small amount of inositol is added thereto.

EXAMPLE 3

Preparation of stabilized lyophilized hepatitis A live vaccine:

Essentially, the stock suspension of hepatitis A live vaccine can beprepared by the method described in detail in CN Patent No. 92114998.Briefly, propagating human fetal lung diploid fibroblast cells infectedwith HAV strain L-A-I derived from human faces, which strain had beenestablished by Dr. Wang Penfu and his colleague in Changchun Instituteof Biological Products, Ministry of Public Health, Changchun, China, inappropriate virus infectious dose in minimum essential medium (MEM)containing 10% fetal calf serum (FCS) at 37° C. for 3 to 4 weeks byserial passaging. When the amount of positive infected cells is morethan 90% as detected by indirect immunoflurescence technique. Thenutrient medium is discarded from culturing vessel, and the residual FCSis washed away by phosphate-buffered saline (BPS). The cultured mediumreplaced by medium 199 without phenol red therein, and the infectedcells are cultivated for a additional 4 to 6 days. After completion ofthe culturing and collecting the infected cells by low-speedcentrifugation, the infected cells are disrupted by means of 3 cycles offreeze-thawing and sonification. Cellular debris is removed bycentrifugation and the supernatant is collected as a stock of thevaccine. The stock material (suspernatant product) will give a positiveresult for antigen by indirect immunoflurescence assay.

After the hepatitis A vaccine is formulated by mixing the stabilizersolution (I) prepared in Example 1 and viral stock suspension preparedas stated above at about 1:1 (v/v) ratio, the resultant vaccineformulation is divided into a small volumes (0.5 ml) into 2 ml glassvial. And then the aliquots of the formulated viral vaccine are placedinto a freeze dryer (model FS150-SS20C, Hull Co., USA) for multisteplyophilization cycle at −40° C. for 4 hours, and then the shelftemperature is gradually increased to about −30° C. and maintainedprimary drying. The shelf temperature is then gradually increased to 32°C. and maintained there for 15 hours to obtain desired lyophilizedstabilized hepatitis A vaccine formulation with a very low moisturecontent.

EXAMPLE 4

Preparation of stabilized lyophilized measles live vaccine:

The stock suspension of attenuated live measles vaccine is prepared inaccordance with the Requirement of Measles Vaccine, Live in ChineseRequirements for Biological Products. The stock material is mixed withthe stabilizer solution (II) prepared in Examples 2 at 1:1 (v/v) ratioto obtain measles live vaccine formulation. The vaccine formulation isprecooled at −40° C. for 5 hours, and then the formulation is subjecteda drying treatment at about −35° C. to 34°0 C. for 14 hours to result inthe lyophilized stabilized measles live vaccine.

EXAMPLE 5

Storage stability testing of lyophilized hepatitis A live vaccine:

The samples of lyophilized hepatitis A live vaccine from different lotsof viral formulation prepared in Example 3 which are stored at 2-8° C.for 3 to 12 months, are ten-fold serially diluted, then the sample of10⁻² to 10⁻⁷ dilution is used for detecting the viral titers every threemonths. The vaccine formulation from the same lot and lyophilized by thesame lyophilization cycle parameters without stabilizer is used as acontrol sample. After adding distilled water for injection to thelyophilized vaccine for reconstitution, the resultant aqueous suspensioncontaining the live virus and stabilizer is subjected to testing forstorage-stability by determinating viral titers (CCID₅₀) usingconventional enzyme linked immunosorbent assay (ELISA) andindirect-immunofluorescence assay (IF). The results of the testingreveal that the 5 lots of samples which were lyophilized in the presenceof stabilizer solution exhibited higher infectious titers in the rangefrom about 6.33 to about 6.50 log CCID₅₀, whereas the 5 lots of controlsamples lyophilized in the absence of stabilizer solution exhibitedremarkably decreased infectious titers in the range from about 1.33 to2.33 log CCID₅₀.

In another experiment, the lyophilized virus samples from the same lotof vaccine formulations were stored at 2-8° C., 25° C. and 37° C.,respectively, and each of the samples were sampled every day andsubjected to testing for storage stability in terms of lowest validstorage periods by detecting the CCID₅₀ values. Live vaccine in the formof aqueous suspension are compared to lyophilized vaccine formulation.

The results are summarized in Table 1 to Table 2 below, respectively.

TABLE 1 Storage stability test of lyophilized live hepatitis A vaccineformulation with stabilizer: Lot number Months of storing at 2-8° C. ofsample 0 3 6 9 12 1  6.50* 6.67 6.67 6.50 6.50 2 6.67 6.50 6.67 6.506.67 3 6.50 6.50 6.33 6.50 6.50 4 6.50 6.67 6.67 6.50 6.33 5 6.33 6.506.50 6.50 6.33 control sample 2.33 1.75 1.50 1.50 1.33 *Infective titersof the virus (log CCID₅₀/ml)

TABLE 2 Comparison of stability of hepatitis A live vaccine: Temperatureof Lowest valid storage period (days) storage Aqueous suspensionLyophilized formulation 2-8° C. 180 360 25° C.  7  90 37° C.  1  7

It can be seen from the results shown in Table 1 and Table 2 as above,that the stabilizer for lyophilized live vaccine of the presentinvention greatly increased thermo-stability measured as the log CCID₅₀,due to the stabilized structure of viral protein and nucleic acid, andeffectively maintains viral potency of the vaccine under the conditionsof increased temperature and osmotic strength.

EXAMPLE 6

The Immunogenicity and Safety Testing of the Lyophilized Hepatitis ALive Vaccine:

The lyophilized hepatitis A live vaccine formulation prepared inaccordance with the method in Example 1 which has been stored at about25° C. for 30 days is intravenously inoculated into healthy rhesusmonkeys (each group comprising 5 animals). Every two weeks the monkeyswere bled for 8 weeks and checked for abnormally elevated serum enzymes(GPT) levels and the titers of anti-HAV antibody. Abnormal elevations ofenzymes (more than 25U/ml) would indicate the presence of hepatitis Adisease in the animals and the presence of antibody would shownprotection (Table 3). In this experiment, a fresh vaccine preparation ininitial state from the same lot but which is unlyophilized and withoutstabilizers therein is used as a control sample. All of the animalsreceived a 10^((6.5)) CCID₅₀ viral infectious dose (1.0 ml of thestock). The results are summarized in Table 3 below.

TABLE 3 Serum GPT abnormal elevation and antibody response of animalsbefore and after inoculation with the lyophilized hepatitis A vaccine.Abnormal elevation anti-HAV of SGPT* anti-HAV IgG Ab** IgM Ab** Lots 0 24 8(w) 0 2 4 8(w) 0 2 4 8(w) 1 0/5 0/5 0/5 0/5 0 60 100 100 0 60 40 0 20/5 0/5 0/5 0/5 0 40 100 100 0 80 40 0 3 0/5 0/5 0/5 0/5 0 60 80 100 060 40 20 4 0/5 0/5 0/5 0/5 0 60 60 100 0 80 20 0 *Serum GPT value ≧ 25U/ml is considered to be abnormal elevation of the enzyme. **The datagiven in the table represent percentage seroconvertion rates from 4animals.

It is can be seen from the results showed in Table 3, that all rhesusmonkeys developed anti-HAV protective antibody and more than 80% ofseroconverted animals also developed IgM anti-HAV at about two weeksafter inoculation. On the other hand, none of the rhesus monkeys hadelevated liver enzymes atributable to the vaccination. All values forthese higher primates were within normal limits. This indicates nobiochemical evidence of hepatitis. These results exhibited comparableimmunogenicity and safety with control samples, and show that thelyophilized live vaccine formulation of the present invention which hasbeen stored for 30 days at ambient temperature still maintains a similarimmunogenicity and safety to its initial state.

EXAMPLE 7

Storage-stability of lyophilized measles live vaccine:

Storage-stability testing of measles live vaccines pro- andpost-lyophilization stored at 2-8° C. and 37° C., respectively, wereperformed in substantially the same manner as described in Example 5.

The results show that, in the presence of stabilizer of the presentinvention, 5 lots of measles vaccine exhibited a slightly decreasedthemostability subsequent to lyophilization, that is less than 0.5 logloss in comparison to the control vaccine in initial state. Further, thelyophilized measles vaccine sample stored at 2-8° C. for 15 months andat 37° C. for 4 weeks had a CCID₅₀ reduced by 0.5 and 1.0, respectively.

1. A stabilized lyophilized hepatitis A live vaccine formulationcomprising prophylactically effective titers of live attenuatedhepatitis A virus and a stabilizer, wherein said live attenuatedhepatitis A virus is prepared from the wild-type HAV, strain L-A-I, andwherein said stabilizer is present in the vaccine formulation at aconcentration sufficient to stabilize the hepatitis A virus against heatinactivation.
 2. A stabilized lyophilized hepatitis A live vaccineformulation according to claim 1, wherein said stabilizer forlyophilized live hepatitis A virus comprises human serum albumin orgelatin or both of them; trehalose; at least one amino acid selectedfrom the group consisting of glutamic acid, aspartic acid, arginine,lysine, and alkali metal salts of any of the foregoing; ascorbic acid;area; mannitol or sorbitol or both of them; and inositol.
 3. Astabilized lyophilized hepatitis A live vaccine formulation according toclaim 1, wherein said stabilizer for the lyophilized live viruscomprises from 0 to 20 grams per liter of human serum albumin, from 5 to10 grams per liter of gelatin, from 50 to 100 grams per liter oftrehalose, from 7.5 to 15 grams per liter of sodium glutamate, from 0.5to 5.5 grams per liter of ascorbic acid, from 5 to 28 grams per liter ofurea, from 2 to 10 grams per liter of mannitol or sorbitol, and from 4to 10 grams per liter of inositol.
 4. A method of preparing stabilizedlyophilized live hepatitis A vaccine formulation according to claim 1,comprising: (a) providing a stock suspension of attenuated liveHepatitis A virus, wherein said live attenuated hepatitis A virus isprepared from the wild-type HAV, strain L-A-I; (b) adding a stabilizersolution to stock suspension of attenuated live hepatitis A virusobtained from step (a) at the ratio 1:1 (v/v) to obtain a live vaccineformulation comprising prophylactically effective titers of liveattenuated hepatitis A virus and a stabilizer for attenuated live virus,wherein said stabilizer comprises gelatine; trehalose; at least oneamino acid selected from the group consisting of glutamic acid, asparticacid, arginine, lysine, and alkali metal salts of any of the foregoing;ascorbic acid; urea; mannitol or sorbitol or both of them; and inositol;and (c) lyophilizing said vaccine formulation obtained from the step(b).
 5. The method according to claim 4, wherein said lyophilizing stepcomprises pre-cooling the vaccine formulation to about −20° C. to −50°C. for about 3 to 6 hours, then drying the live vaccine formulation bygradually increasing the temperature to at least 32° C. in alyophilizer.
 6. The method according to claim 4, wherein said stabilizercomprises from 0 to 20 grams per liter of human serum albumin, from 5 to10 grams per liter of gelatin, from 50 to 100 grams per liter oftrehalose, from 7.5 to 15 grams per liter of sodium glutamate, from 0.5to 5.5 grams per liter of ascorbic acid, from 5 to 28 grams per liter ofurea, from 2 to 10 grams per liter of mannitol or sorbitol, and from 4to 10 grams per liter of inositol.
 7. The method according to claim 4,wherein said lyophilizing step comprises pre-cooling the vaccineformulation to about −20° C. to −50° C. for about 3 to 6 hours, thendrying the live vaccine formulation by gradually increasing thetemperature to at least 32° C. in a lyophilizer; and wherein saidstabilizer comprises from 0 to 20 grams per liter of human serumalbumin, from 5 to 10 grams per liter of gelatin, from 50 to 100 gramsper liter of trehalose, from 7.5 to 15 grams per liter of sodiumglutamate, from 0.5 to 5.5 grams per liter of ascorbic acid, from 5 to28 grams per liter of urea, from 2 to 10 grams per liter of mannitol orsorbitol, and from 4 to 10 grams per liter of inositol.
 8. A stabilizerfor lyophilized live virus, wherein said stabilizer comprises gelatin;trehalose; at least one amino acid selected from the group consisting ofglutamic acid, aspartic acid, arginine, lysine, and alkali metal saltsof any of the foregoing, ascorbic acid; urea; mannitol or sorbitol orboth of them; and inositol.
 9. A stabilizer according to claim 8,wherein said stabilizer comprises from 0 to 20 grams per liter of humanserum albumin, from 5 to 10 grams per liter of gelatin, from 50 to 100grams per liter of trehalose, from 7.5 to 15 grams per liter of sodiumglutamate, from 0.5 to 5.5 grams per liter of ascorbic acid, from 5 to28 grams per liter of urea, from 2 to 10 grams per liter of mannitol orsorbitol, and from 4 to 10 grams per liter of inositol.
 10. A stabilizeraccording to claim 8, wherein said lyophilized live virus is selectedfrom the genus Enterovirus.